Project Summary Clinical management of obstructive lung diseases such as asthma and chronic obstructive pulmonary disease (COPD) relies heavily of drugs that are either agonist or antagonists of G protein-coupled receptors (GPCRs). This is because GPCRs control important airway cell functions (contraction, growth, cytokine/chemokine production, chemotaxis, and permeability) that become dysregulated in disease. Unfortunately, these drugs have limitations with respect to both efficacy and safety, and many patients experience suboptimal control. We propose to identify new, more effective ligands that target the M3 muscarinic acetylcholine receptor (M3 mAChR) as a potential COPD therapy, by embracing the emerging concept of biased agonism pharmacology. Biased agonism pharmacology is based on the discovery that GPCRs can assume multiple conformations that transduce qualitatively different signals, and ligands can ?tune? GPCRs to bias signaling to one pathway or another. We propose to discern qualitative signaling properties of the M3 mAChR and determine how canonical (G protein-dependent) versus non-canonical (arrestin-dependent) signaling regulates airway smooth muscle (ASM) functions that are often dysregulated in COPD. We have discovered both small molecule orthosteric ligands as well as a peptide that can bias M3 mAChR signaling toward the arrestin signaling pathway. Preliminary data also strongly suggest arrestin-dependent signaling mediates therapeutically beneficial ASM functions. Lead agents in hand as well as those that emerge in focused screens will be tested in cell- and tissue-based studies of ASM signaling and function, and ultimately in an in vivo model of COPD to discern the utility of these drugs as COPD therapeutics. Three Aims are proposed. Aim 1 will employ genetic and molecular strategies, including use of mice with arrestin subtype genes ablated, to establish the regulatory effects of arrestins on M3 mAChR-mediated functions (contraction, growth, and synthetic functions) in airway smooth muscle. Aim 2 will employ semi-high throughput screening systems, ASM signaling and functional assays, and novel engineered M3 receptors to fully characterize those biased agents in hand and to discover additional biased ligands and peptides. Aim 3 will test our lead biased M3 ligands in an in vivo model of COPD to verify the ability of a biased pharmacology approach to manage the disease. The impact of the proposed studies lies in their ability to significantly advance the science underlying, and ultimate clinical application of, biased agonism pharmacology.